1. Technical Field of the Invention
The present invention relates to the field of liquid chromotagraphy. In particular, the present invention is directed to a an improved apparatus for use in obtaining the high and uniformly pressurized slurry beds employed in present liquid chromatography columns, as well as a method for achieving such uniformly packed columns.
2. Description of Background Information
Chromatography is a separation method whereby individual chemical compounds which were originally present in a mixture are resolved from each other by the selective process of distribution between heterogeneous phases. The distribution of chemical species to be separated occurs in a dynamic process between a mobile phase and a stationary phase. The stationary phase, or the column packing material, usually has a relatively large surface area through which the mobile phase is allowed to flow. The chemical nature of the stationary phase exercises the primary control over the separation process. The greater the affinity of a particular chemical compound for the stationary phase, the longer it will be maintained in the system. The mobile phase can be either gas or liquid; correspondingly, the methods are referred to as gas chromatography and liquid chromatography.
Chromatography has been used primarily as a separation and isolation method, and is often intended to separate many mixture components in a single-step procedure. Chromatographic methods can be applied to a very wide concentration range; from commercial scale quantities (gram-kilogram range) all the way to analytical determinations of the order of 10.sup.-9 gram. Chromatographic separations are based upon the physicochemical principals of adsorption and partion, and, conversely, these and related fundamental physicochemical phenomena can be studied by high precision chromatography of model systems.
In liquid chromatography, the mobile phase may be perculated through the column at atmospheric pressure, by means of gravity, or under more contemporary procedures, through the pressure generated by smaller and smaller particles of the packing media. High pressure pumps which can generate up to several thousand atmospheres of inlet pressure are often used.
Thus, high pressure liquid chromatography (HPLC) is a process of separating complex mixtures of chemicals by passing a solution of the chemicals through a stationary column packing material and under a certain degree of pressure. Preparative high pressure liquid chromatography is a process in which one applies a mixture of a compound in solution and in its mobile phase where, by virtue of the selection of the components of the column packing material, the column will retain the various and select components of the mixture for that length of time which causes its select components to be eluted discretely for separate collection as individual components and as purified elements. In preparative HPLC the separation and purification is in larger quantities than in analytical HPLC.
Increasingly, preparative liquid chromatographic separations are being carried out using porous, silica based, or polymeric micro-particulate column packings. These materials offer the advantages of increased resolution and improved overall performance in chromatographic separations.
The field of liquid chromatography has long been utilized in the fields of chemistry, biochemistry and biotechnology. Generally, in practicing the art of chromatography, a slurry is packed within a preparative column under pressure. It has been an ongoing problem in this field to obtain a fine and uniform degree of compactness of the slurry bed within the column as the diameter and length of the preparative column increases in order to obtain the most efficient and effective use of a column during the performance of the chromatographic process. One of the major problems of the prior art packing of liquid chromatography columns has been the formation of bubbles or soft and mushy pockets within the packed bed of the column.
More recently, the field of liquid chromatography has grown to include what is known as high pressure liquid chromatography (HPLC), wherein the columns to be packed are employed in analytical semi-preparative or process columns for use in chromatography. The columns used in chromatographic analysis are generally fabricated of metal, such as stainless steel, and function to separate various chemicals (for analysis or use) that pass through the columns at pressures that range from up to 2000 to 4000 psi. By the use of a liquid chromatography column, the chemicals passed through the absorbent bed are separated into their constituents so that the various desired elements can be obtained, or an analysis of the chemical passing through the column can be performed. A critical feature in ensuring the proper operation of such columns relates to the degree and uniformity of compactness of the packed absorbent bed contained within the column. This slurry packing must be maintained at a very high pressure, and at a uniform density, in order to achieve the most efficient and effective results during the chromatographic procedure.
The present invention relates to the configuration and structure of columns for use in preparative liquid chromatography. The present invention also relates to an apparatus for ensuring the uniformity of slurry and pressure applied to such a column while at the same time minimizing and/or preventing leakage of particles past the pressure applying mechanism. The slurry compressor of the present application facilitates obtaining a homogeneous absorbent packed bed in liquid chromatography columns, thus improving the efficiency of the columns and increasing the accuracy of the results obtained thereby. The particularly designed and styled compressor of the instant application is particularly designed to eliminate the leakage of particles past the compressor while at the same time enabling very high and uniform pressures and densities to be achieved. The present invention also relates to an adapter or tool which facilitates and/or enables insertion of the inventive slurry compressor into the column without damaging the compressor or the sealing members carried thereby. The present invention also relates to a method of mixing the slurry prior to packing to enhance its separating qualities.
The instant slurry compressor is an improvement over the compressor disclosed in U.S. Pat. No. 4,675,105 (MARTIN et al.) entitled "System for Obtaining a Homogeneous Absorbent, Bed in a Chromatographic Column".
The present invention also relates to a handled straight adapter for use between two chromatographic columns that facilitates packing of the working column from an attached reservoir column. The instant handled straight adapter is an improvement over the adapter disclosed in U.S. Pat. No. 4,719,011 (SHALON et al.) entitled "High Pressure Liquid Chromatography Columns".
One very successful solution to the problem of achieving high and uniform pressures and densities within liquid chromatography columns is set forth in the above-mentioned MARTIN et al. patent. In that patent, a slurry compressor is disclosed for obtaining a homogeneous absorbent bed in a liquid chromatographic column. The compressor includes a solid, shaped compressor member having external dimensions only very slightly less than the internal dimensions of the column in which the bed of particles being compressed is contained. The compressor includes an innermost portion which, in use, is in contact with the particles within the column and an outermost portion integrally connected to the innermost portion. The innermost portion is a solid segment while the outermost portion is formed with a hollowed interior forming a walled structure extending rearwardly from the solid portion. The walled structure of the outermost portion is shaped to flare slightly outwardly from the inner portion in order to tightly and sealingly contact the inner wall surface of the associated reservoir column. Thus, the flared wall portion acts as a seal, preventing the slurry from leaking past the compressor element.
While the above mentioned prior art slurry compressor has functioned well, a need nevertheless exists for an improved sealing mechanism for preventing leakage of slurry past the compressor, particularly at the very high pressures used in modern chromatographic systems. The present invention solves this problem by providing a slurry compressor designed in such a manner so as to prevent leaks of slurry therepast within the column.
Also provided in the present invention is a tool and adaptor element that cooperates with the slurry compressor and with the column for enabling the slurry compressor to be quickly and efficiently inserted into the liquid chromatographic column without damaging any portion thereof.
The present invention thus relates to an improved slurry compressor having an improved resilient sealing means, to an adaptor tool for inserting the compressor into the chromatographic column without damaging any portion thereof, to a method of mixing the slurry prior to compression thereof, as well as to an adaptor element with outwardly extending handles.
The efficiency of the column used in liquid chromatography is very strongly influenced by features such as the length and/or width of the column. Thus, to be able to separate a large variety of chemical compounds it has in the past been necessary to maintain in storage, have available or purchase a large variety of columns of various lengths and/or widths. This of course has been extremely cumbersome, not to mention expensive and inefficient. A very successful solution to the problem of the non-adaptivity of the various columns to a wide variety of chemical compounds was proposed by the aforementioned SHALON et al. patent. Therein, a modular chromatographic column was proposed. According to the disclosure of the above patent, columns of varying length and width can be coupled together through the use of a variety of adapters. The system disclosed is quite easy to assemble because all of the components thereof are modular and are very convenient to handle. Thus, a large stock of different lengths and widths of columns is no longer needed. Practice of the art of liquid chromatography has been simplified and thus greatly enhanced through the use of a relatively limited number of modular components.
The above-mentioned modular liquid chromatography system has been quite successful in providing a wide range of column widths and lengths by virtue of the use of various types of adapters, such as straight adapters for connecting columns of equal diameter and conical or tapered adapters for connecting columns having different diameters. However, in the use of these adapters a problem in removing the adapter from the column itself occasionally arose. The present invention solves this problem by providing an adapter having handles integrally attached thereto to facilitate removal of the adapter from the column. After a bed has been pressurized and packed within a column and an associated reservoir column, and the reservoir column is removed prior to the use of the packed working column, the bed has tended to expand upwardly and outwardly away from the working column. As the pressurized bed expands upwardly, as a result of the removal of pressure therefrom, it can become damaged, e.g., by breakage. The present invention solves this problem by providing an adapter having handles attached integrally thereto to facilitate restraining the upward expansion of the pressurized slurry bed from the working column. This permits removal of the reservoir column from the working column and removal, as by a wire knife or other means, of the extra packing media from the working column as it expands therefrom. The use of the instant handled straight adapter which is used to connect both columns prevents damage to the prepared packed bed within the working column.
The present invention also relates to an improved straight adapter for coupling two column portions together. The improved adapter of the present invention is provided with handles so that during use (i.e., filling and packing of the column) the column can be stabilized by being attached to a support structure via the handles attached to the adapter, and upon disassembly of the columns, because of the handles provided on the adapter, the prepared packed column bed is not damaged.
Since the filling, assembly, and pressurization (packing) of the column takes some period of time, the filling material (slurry mixture) often tends to undesirably settle out during the filling process. According to a further feature of the present invention, a method for filling and preparing chromatographic columns is set forth that eliminates such problems and enables the columns to be filled in such a manner that the contents of the slurry bed therein remain uniform. This has made the slurry packed columns much more efficient and effective in being utilized for preparatory chromatography.
The double sealed slurry compressor with the flared end up is inserted on the top of the reservoir column with the aid of the compressor adapter. Once the compressor is in place the adapter is removed form the reservoir column. The top of the reservoir column is now sealed with a large O-ring and an end plate. The end plate is mounted securely with bolts and nuts. This endplate will later be connected to the packing instrument.
According to a most significant feature of the present invention, after the working and reservoir columns have been mounted together with the inventive straight handled adapter mounted therebetween, the mixed slurry is then poured into the assembled columns. The assembly is not filled totally, thus a substantial amount is left empty, which will create an air bubble in the assembly. The working column is now sealed with a large and small O-ring, a bed support frit, a disperser frit, and an end plate and tightened with bolts and nuts. The assembled columns are now sealed and shaken or mixed so that the contents (i.e., the slurry mixture) are mixed so that the air bubbles contained within the column will serve as a mixture device to maintain the homogeneous nature of the slurry and prevent settling out of the slurry components prior to the application of pressure to the slurry filled assembly. Immediately thereafter, the slurry can be pressurized to a uniform and high pressure and density by applying pressure to the instant improved double sealed slurry compressor, which is positioned on top of the reservoir column which is attached to the packing instrument. After the system has been pressurized and allowed to equilibrate for a period of time, the procedure of removing the reservoir column from the working column can be performed only after the pressure is released. First, most of the nuts and bolts connecting the straight adapter and reservoir column to the working column are removed, and the remaining ones are loosened, so that a cutting wire can be inserted to cut the slurry between the columns with a cutting wire. The reservoir column is then very carefully removed while a stainless steel plate is placed on top of the packing media within the handled straight adapter (not yet removed from the working column). The plate and handled straight adapter are then held in place securely while the attaching means, that attach the straight adapter to the support structure of the column, are loosened. The straight adapter can then be removed making sure not to destroy the integrity of the packed bed. After this, the working column is sealed by attaching thereto the end plate, various frits, sealing O-rings and other mechanisms by means of nuts and bolts, the nuts and bolts are tightened, and the packed column is ready for use.